RESEARCH ARTICLE 3887

Development 137, 3887-3898 (2010) doi:10.1242/dev.055871 © 2010. Published by The Company of Biologists Ltd Hedgehog targets in the Drosophila embryo and the mechanisms that generate tissue-specific outputs of Hedgehog signaling Brian Biehs, Katerina Kechris*, SongMei Liu and Thomas B. Kornberg†

SUMMARY Paracrine Hedgehog (Hh) signaling regulates growth and patterning in many Drosophila organs. We mapped chromatin binding sites for Cubitus interruptus (Ci), the that mediates outputs of Hh signal transduction, and we analyzed transcription profiles of control and mutant embryos to identify genes that are regulated by Hh. Putative targets that we identified included several Hh pathway components, mostly previously identified targets, and many targets that are novel. Every Hh target we analyzed that is not a pathway component appeared to be regulated by Hh in a tissue-specific manner; analysis of expression patterns of pathway components and target genes provided evidence of autocrine Hh signaling in the optic primordium of the embryo. We present evidence that tissue specificity of Hh targets depends on transcription factors that are Hh- independent, suggesting that ‘pre-patterns’ of transcription factors partner with Ci to make Hh-dependent gene expression position specific.

KEY WORDS: Drosophila, Hedgehog, Cubitus interruptus

INTRODUCTION Pleiotropic phenotypes result from loss or inactivation of Hh Hedgehog (Hh) is a secreted signaling protein that regulates the pathway components, suggesting that the pathway is similarly growth and patterning of many organs. First identified because of constituted in the affected tissues (reviewed by McMahon et al., its roles in Drosophila embryonic and imaginal disc development, 2003). Ci is an essential core pathway component (Alexandre et al., it is now understood to be essential to most organs in Drosophila 1996; Forbes et al., 1993) and is stabilized by Hh signal and higher vertebrates. Despite its systemic importance, Hh transduction (Aza-Blanc et al., 1997; Ruel et al., 2003). Expression signaling is local and is dependent upon Hh that is expressed and of ptc (Forbes et al., 1993; Ingham, 1991; Tabata and Kornberg, secreted by discrete sets of cells in each tissue that it regulates. 1994) and roadkill (rdx) (Kent et al., 2006; Zhang et al., 2006) are In each context, organ-specific programs of gene expression, also hallmarks of Hh action. Many of the pathway components are morphogenesis and cell-cycle regulation depend upon Hh evolutionarily conserved in higher vertebrates and most of these regulation. The mechanism that endows Hh signaling with tissue homologs have been found to play similar roles in the specificity has not been fully elucidated. corresponding Hh pathways (reviewed by Varjosalo and Taipale, Hh signaling has been most thoroughly analyzed in the 2008). The deep homology extends to the three Gli transcription Drosophila wing imaginal disc, where Hh is expressed in posterior factors (Orenic et al., 1990), homologs of Ci that share a consensus compartment cells. Signaling to adjacent anterior cells begins when binding sequence (TGGGTGGTC) (Hallikas et al., 2006; Kinzler Hh engages the participation of two membrane proteins in target and Vogelstein, 1990; Pavletich and Pabo, 1993). cells: Patched (Ptc) and Smoothened (Smo). Hh activates Smo As with all signaling systems that employ a common mechanism (Stone et al., 1996; Taipale et al., 2002) and initiates the of signal transduction to regulate a variety of target tissues, the transformation of a complex of proteins that includes an inactive question arises of how tissue-specific responses are induced. For the form of the transcription factor Ci (Jia et al., 2003; Lum et al., Hh pathway, the question of specificity also applies to CiAct and 2003; Ruel et al., 2003). Ci mediates most, and perhaps all, of the CiRep, which regulate expression of targets in the on and off states, output of the Hh pathway (Alexandre et al., 1996; Méthot and respectively. Analysis of the imaginal disc enhancer of Basler, 2001). In the absence of Hh, cleavage of Ci generates a decapentaplegic (dpp) revealed that both CiAct and CiRep can bind truncated peptide that functions as a transcriptional to, and regulate, the same binding site sequence (Müller and Basler, (CiRep) (Aza-Blanc et al., 1997). The presence of Hh reduces CiRep 2000). However, we do not yet know whether such shared access is and enhances production of a transcriptional activator form (CiAct) a feature of all Ci binding sites and, if it is, how the distinct activator (Aza-Blanc et al., 1997; Méthot and Basler, 1999). and repressor functions are realized in the context of their common sites. Possible mechanisms include differential affinity, input from linked cis-regulatory elements or participation of co-factors (Müller and Basler, 2000). Although support for the latter might come from Cardiovascular Research Institute and Department of Biochemistry and Biophysics, the observation that full activation of the dpp heldout enhancer University of California, San Francisco, CA 94143-2711. requires both Hh signaling and the wing ‘selector’ protein Vestigial *Present address: Colorado School of Public Health, University of Colorado Denver, (Hepker et al., 1999), the mechanisms that determine tissue-specific 13001 E. 17th Place B-119, Aurora, CO 80045 activation of other Hh targets remain unidentified. Recent studies †Author for correspondence (tkornberg(at)ucsf.edu) that used chromatin binding to identify target sequences for mouse

Accepted 16 September 2010 Gli1 and Gli3 proteins in neural tissues and whole limb buds, DEVELOPMENT 3888 RESEARCH ARTICLE Development 137 (22) respectively (Vokes et al., 2007; Vokes et al., 2008), identified many transgenes. DNA (2.5 mg) was ethanol-precipitated, digested with DpnI and novel Gli-responsive cis-regulatory elements. Characterization of ligated to annealed adaptor oligos for 2 hours at 16°C (5Ј-CTAA - these elements led to the conclusion that the Gli1 activator and the TACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3Ј and Gli3 repressor recognize and regulate common sequences. 5Ј-TCCTCGGCCG-3Ј). After heat inactivation, the ligation mixture was The number of known Hh targets in Drosophila is small. The digested with DpnII, which cuts unmethylated DNA. PCR amplification of work described here was undertaken to obtain a better understanding Dam-methylated DNA was carried out using the PCR Advantage Mix (Clontech) and the primer 5Ј-GGTCGCGGCCGAGGATC-3Ј. Labeling of targets and to investigate the mechanism of Ci action and and hybridizations were performed by NimbleGen Systems (Reykjavík, specificity. We employed expression array assays to identify genes Iceland). The amplified methylated fragments were labeled with Cy3 or that have Hh-dependent expression in embryos, and chromatin- Cy5 in a dye-swap configuration to eliminate bias and were hybridized to binding assays to identify genes linked to genomic regions that are NimbleGen Systems’ whole genome tiling arrays consisting of 375,000 recognized by CiAct and CiRep. Putative targets were selected that 60mer probes spaced ~300 bp apart (Choksi et al., 2006). Three replicates bind both Ci forms and are Hh-dependent. Characterization of of control (Dam alone) and experimental (DamCiAct and DamCiRep) several novel targets highlighted two key aspects of Hh signaling. samples were analyzed. Raw intensity data were analyzed as described We found evidence for autocrine signaling and also found that, with below. the exception of several genes that encode core components of the All analyses were performed in R v2.7.1 (R Development Core Team, Hh signal transduction pathway, all targets are expressed in restricted http://www.R-project.org/) unless otherwise stated. At each tiling array feature, log ratios between the experimental and control samples were domains within Hh-responsive tissues. For Ecdysone-inducible gene calculated and normalized using ‘limma’ (Smyth, 2005), applying ‘loess’ L2 (ImpL2), a novel Hh-regulated gene that is expressed in the and ‘Aquantile’ for normalization within arrays and between arrays, tracheal primordium, we show that expression is dependent upon the respectively (Wormald et al., 2006). To predict binding regions, each transcription factor Trachealess (Trh), expression of which is not feature was tested for significant positive intensity using a one-sided t-test. dependent upon Hh, and that ImpL2 expression is restricted to only Because sample size was small (n3), information was pooled from all a portion of the cells that express Trh. Expression of this Hh target features to obtain more stable variance estimates for the t-statistic using the is, therefore, specified by the combined activities of Hh-dependent limma package. To identify genomic regions of high signal intensity, P- Ci and Hh-independent Trh. values for consecutive features were ‘averaged’ using meta-analysis based on combining P-values (for details, see Kechris et al., 2010). Because of MATERIALS AND METHODS the large number of significance tests, P-values were corrected for false Fly stocks and crosses discovery rate (FDR) control (Benjamini et al., 2001) with window size set Homozygous null mutant embryos lacking ptc or hh function were at four. Binding regions were created by scanning features as they are generated from the following alleles: hh13c, hhAC, ptcB98 and ptc13C. ci-null ordered along each chromosome. If a feature had an adjusted P-value Rep Act embryos were generated from a cross of ciRES (Méthot and Basler, 1999) below the set FDR cutoff (0.02 for DamCi ; 0.001 for DamCi ), a new in a ci-null background (ci94/ci94). smo germline clones were generated binding region was formed. The next feature passing the cutoff in the linear from a cross of y w hs-FLP/+; smoQ FRT40A/CyO females to +/Y; OvoD1, chromosome was then evaluated. This procedure was continued in a step- FRT40A/CyO males (Chou and Perrimon, 1996). Embryos and first instar wise fashion. progeny were heat shocked daily at 37°C for 1 hour prior to eclosion. UAS- m1-m4 Act Overlap of DamID protected regions Ci (Ci ) was obtained from S. Smolik (Oregon Health and Science The overlap binding regions were evaluated using two metrics: (1) overlap University, Portland, OR, USA) and the Dam alone transgenic line was of set of all proximal transcripts and (2) base pair overlap between binding obtained from S. Parkhurst (Fred Hutchinson Cancer Research Center, regions. To compare these values with chance occurrence, binding regions Seattle, WA, USA). for CiRep were generated randomly fifty times based on length distribution In situ hybridization and immunostaining and number of binding regions. Each random set of binding regions was Act Digoxygenin-labeled anti-sense RNA probes were hybridized to whole- compared with the Ci binding region, and the two overlap metrics were mount embryos (O’Neill and Bier, 1994). Antibodies used for calculated. To obtain gene and base pair overlap, enrichment scores of immunostaining were: rat anti-2A1 (CiFl; R. Holmgren, Northwestern original values for overlap metrics were divided by values based on an University, Evanston, IL, USA; 1:2000), mouse anti-Patched (I. Guerrero, average of 50 randomizations. This analysis was also repeated by Universidad Autonoma de Madrid, Madrid, Spain; 1:200), mouse anti- performing the binding region randomizations on each of the 21 Fasciclin 2 (Y. N. Jan, University of California, San Francisco, CA, USA; transcription factors analyzed in a previous study (MacArthur et al., 2009) Act 1:1000) and rabbit anti--galactosidase (Y. N. Jan; 1:5000). The signal was and comparing those with the Ci binding regions. We found extensive visualized with Alexa 488-conjugated secondary antibodies (Molecular overlap to the same regions identified by MacArthur and colleagues. Given Probes) or the ABC Vectastain Kit (Vector Laboratories) as described the extensive overlap within the MacArthur data set for the many different previously (Aza-Blanc et al., 1997). transcription factors that they and others have profiled, these binding sites appear to be detected by several contrasting techniques and are of uncertain DamID constructs significance. N-terminally fused DamID constructs were generated by PCR amplification of BglII-XbaI fragments of Ci76 (Aza-Blanc et al., 1997) and Incidence and frequency of Ci motifs Cim1-m4 (S. Smolik) using the forward primer 5Ј-TAAGATCTTATG - The sequences of the DamID binding regions were extracted from FlyBase GACGCCTACGCGTTACCTAC-3Ј and reverse primers 5Ј-TAATCT - v4.0, and occurrence of TGGGTGGTC and reverse complement were AGAGTCTGCCACGTCCACGTCATCGT-3Ј for Ci76 and 5Ј-TAATCT - counted (Perl script v5.10.0). The rate of this motif, as well as the AGACTGCATCATTTGAAGGTATCTATTTTCC-3Ј for Cim1-m4. PCR occurrence of ‘relaxed consensus’ sequences, in binding regions and products were digested with BglII and XbaI and ligated to pNDamMyc genome were calculated by dividing counts of motif by the total length of (B. van Steensel, Netherlands Cancer Institute, Amsterdam, The binding regions or genome, respectively. Enrichment was the rate of motif Netherlands). DamCi fusion cassettes were subcloned into pUAST. occurrences in the peak set divided by the rate in the genome. P-values for enrichment assumed a Poisson distribution. The strict consensus DamID analysis (TGGGTGGTC) occurs 2.5 and 2.4 times more frequently in CiAct and DamID probes were prepared following the protocol described at CiRep binding sites, respectively, than would be expected by chance http://research.nki.nl/vansteensellab/damid.htm. Genomic DNA was (P4ϫ10–24 and 7.2ϫ10–8, respectively). The relaxed degenerate Ci motif Act isolated from 2- to 6-hour-old embryos containing DamCi or Dam (YGSGDGGNC) occurs 1.2 and 1.3 times more frequently in Ci and DEVELOPMENT Targets of Hedgehog signaling RESEARCH ARTICLE 3889

CiRep binding sites (P1.4ϫ10–9 and 4.7ϫ10–11, respectively). The clusters (GSE24024). The gene expression array data (GSE24038) are both the of the degenerate binding motif YGSGDGGNC in the vicinity of Hh log2-transformed ratios and fluorescent intensities for 14 experiments targets ptc, rdx, hh, wg, en, drm, odd and ImpL2 (see Table 2 in the comparing expression in mutant embryos with control siblings. GSE24055 supplementary material) are depicted in Figs 1, 3 and 6 with the presence includes all of the deposited data as one superseries. of clusters of degenerate Ci recognition motifs shown as red boxes. The motif is based on identified Ci recognition motifs (Müller and Basler, 2000; RESULTS Sasaki et al., 1997; Von Ohlen et al., 1997) as well as binding affinities of Drosophila Ci for random nucleotides at each position of the Gli consensus Specificity of Ci binding to the TGGGTGGTC (Hallikas and Taipale, 2006). Sequence coordinates refer genome to Drosophila genome R5.29. The clusters were identified using We used the DamID technique (van Steensel et al., 2001) to locate Flyenhancer software (Markstein et al., 2002), which scans the genome for regions bound by Ci in the Drosophila genome. This method clusters of motifs. We set the following parameters: at least two matches directs DNA methylation to sites of sequence-specific binding by to the motif pattern (YGSGDGGNC) within 600 bp, or at least one of the expressing a fusion protein composed of a transcription factor and ten known Ci recognition sites within 1000 bp. DNA adenine methyltransferase (Dam), and employs hybridization De novo motif analysis to DNA microarrays to detect genomic fragments for which In addition to word searches of the Ci motifs, two programs were used to methylation confers protection from endonuclease digestion. We Act query the peaks associated with the 52 high confidence genes. All peaks generated two DamCi fusion proteins: a constitutive Ci of Dam neighboring the 52 genes (91 peaks, for a total of 504,561 bp) were (DamCiAct) fused to the N terminus of a Ci mutant that activates extracted and masked for repeats using the RepeatMasker tool Hh targets independently of Hh signaling (Chen et al., 1999), and (http://www.repeatmasker.org/) that identifies interspersed repeats and low Dam fused to the N terminus of Ci76 (DamCiRep) (Aza-Blanc et complexity DNA sequences. The masked sequences were then scanned for al., 1997). Examination of wing phenotypes after expression at repeating patterns using default options for the MEME software high levels in wing discs (MS1096GAL4) revealed that CiAct and (http://meme.sdsc.edu) and the oligo-analysis tool at the RSA Tools website CiRep functionality was preserved in the respective fusion proteins (http://rsat.ulb.ac.be/rsat/). MEME found some local repeats (e.g. (not shown). CACACACA) despite repeat masking. The RSA analysis was more To identify Ci targets, methylated DNA fragments were collected successful: of the 35 most significant oligonucleotide sequences that were Act Rep over-represented (occurrence E-value<10–8), most could be clustered by from stage 10-11 embryos expressing DamCi or DamCi . At similarity using the RSA pattern assembly tool into seven motifs of at least this stage, organogenesis is active and Hh signaling activity is three sequences. One included a portion of the Ci consensus site required in many areas. Three replicates were analyzed for each TGGTGGT. Other motifs included GTCCTGC, CTGCTGCC, GTCCTTG, construct; Dam alone embryos served as a control for non-specific CGGCGCG, TTTGTTT and TATTTA. We cannot distinguish whether methylation. Expression was driven by the minimal Hsp70 these sequences are variations of the Ci consensus site or are alternative promoter of the UAS vector without heat shock. We detected 2438 regulatory sequences. Otherwise, these searches, in general, identified little protected regions bound by DamCiAct and 1743 for DamCiRep (see of note. Table S1 in the supplementary material). Median protected region Act Rep Expression array analysis length was 2133 bp for DamCi and 993 bp for DamCi . The RNA was isolated, amplified and labeled as described previously (Klebes differences in frequency and length might reflect differences in and Kornberg, 2008). Data were processed with Cluster and Treeview (Eisen biological activity of the CiAct and CiRep constructs, different levels et al., 1998), and Genepix PRO (Axon Instruments). Normalization for of expression, different affinities or other factors, but, irrespective cluster analysis was carried out with NOMAD 2.0 (University of California, of cause, we assume that this method has a high false positive rate San Francisco, USA). Genes chosen had combined median intensity of >300 (see Materials and methods for further discussion), as do other above background (both channels) in >80% of experiments, and above a related techniques that monitor chromatin binding (Vokes et al., threshold of 1.4 (0.55 of the log2-transformed ratios). The fold-change and 2008; Zeitlinger et al., 2007). To establish the criteria that cluster selection procedure identified 147 transcripts. Using a post hoc FDR distinguish functional sites from false positives, we assessed four evaluation, ~75% of the genes represented by these transcripts were Act Rep controlled at an FDR of 0.1 and 88% were controlled at a FDR of 0.2 based properties: (1) common binding by Ci and Ci , (2) presence of on P-values from a two-sided t-test across all experiments using transcripts Ci binding motifs, (3) linkage to known Hh targets and (4) Hh with at least three non-missing values. dependence of expression levels of linked genes. To assess the degree of similarity in expression values for array Available evidence indicates that CiAct and CiRep can bind to and replicates, Pearson’s correlation coefficient and a concordance percentage function at common sites (Müller and Basler, 2000), and a were calculated between each pair of replicates within a group and between significant number of the DamID-protected regions are shared. 729 replicates across groups. Missing values were removed from both analyses. out of 2439 (30%) of the DamCiAct-protected regions overlapped Higher correlations (positive) or concordance percentages, with a with DamCiRep-protected regions, with a median overlap of 1231 maximum of 1.0 and 100%, respectively, indicate that the arrays are bases; 75% of the overlaps were between 383 and 2147 bases. The generally in agreement. Although there was some variability for replicates within a group (e.g. for the Smo group, one of the lowest correlation values base pairs that overlapped were 8.6 times the number expected by was ~0.3 between two of the replicates), the median correlation and chance. We do not understand the basis for extensive regions of concordance percentage were higher between replicates within the same nuclease protection but simply assume that the location and size of group and were lower between different groups (see Table S3 in the each region correlates in some way with the presence, number and supplementary material). For example, the correlation was 0.62 between affinity of binding sites, and perhaps with local chromatin replicates of the hh arrays whereas the median correlation with other arrays conformation. Irrespective of the underlying mechanism, we was 0.2. analyzed the genome in the vicinity of regions bound by DamCiAct Rep Data access or DamCi to identify transcription units that contain or have Data for the DamID and expression arrays are in the GEO database. adjacent upstream or downstream binding sites for both. The Results from DamID are the normalized log2-transformed ratios of number of transcripts found that contain or are proximal to both Act Rep DamCi/DamAlone signals. The data include three biological replicates for DamCi and DamCi binding regions was 2108. The correlation Act Rep both DamCi / DamAlone (GSE23999) and DamCi / DamAlone of the transcripts that are associated with both binding regions was DEVELOPMENT 3890 RESEARCH ARTICLE Development 137 (22)

Fig. 1. Ci binds at known targets of Hedgehog signaling in Drosophila. (A-F)Averaged log2- transformed ratios of DamCiAct/Dam alone (orange vertical bars) and DamCiRep/Dam alone (blue vertical bars) at statistically relevant regions of the genome (x-axes) for patched (ptc; A), wingless (wg; B), roadkill (rdx; C), atonal (ato; D), hedgehog (hh; E) and engrailed/invected (en/inv; F) genes, all of which had a significant DamID signal. Transcription units are indicated below. Red boxes indicate clusters of Ci binding motifs with at least two motifs matching the consensus YGSGDGGNC within a 600 bp window (for coordinates and sequence of the motifs see Table S1 in the supplementary material). Green boxes (A,D) indicate known enhancers (Forbes et al., 1993; Sun et al., 1998).

statistically significant (1.4ϫ that expected by chance; P-value to distinguish between bona fide binding sites and background, but <2.2ϫ10–16, Fisher’s test) and they represented 67% of DamCiRep that these statistics, as well as the correlation with known target (2108 out of 3160) and 54% of DamCiAct (2108 out of 3939) genes described in the following section, suggest that the presence binding region-associated transcripts. of consensus sequence motifs is a positive indicator for functional We examined the DamCiRep peaks to locate sequences that binding. conform to known Ci binding preferences. Previous work defined Next, we asked if known targets of Hh signaling were among the a consensus binding sequence for the Gli and Ci proteins 2108 genes linked to DamCiAct and DamCiRep peaks. Binding (TGGGTGGTC) (Hallikas and Taipale, 2006; Kinzler and regions for DamCiAct and DamCiRep were detected within 7 kb of Vogelstein, 1990; Pavletich and Pabo, 1993), as well as variants at the transcription units of 27 out of 30 known targets (Table 1). Plots human ptc (PTCH1 – Human Gene Nomenclature database) of DamID protection at five representative targets, ptc, wg, rdx, (Kinzler and Vogelstein, 1990), Drosophila wg (Von Ohlen et al., bagpipe (bap) and atonal (ato), are shown in Fig. 1. At ptc, a 35 1997), Drosophila dpp (Müller and Basler, 2000) and mouse Foxa2 kb DamID-protected region included most of the transcription unit (Sasaki et al., 1997). Probing the Drosophila genome for these as well as ~20 kb upstream. Six Ci consensus binding sites were in sequences (YGSGTGGHC and HDSSHGVHS) identified many the protected region; the sites in the upstream sequence have been sites, but their incidence in the DamID peaks was not significantly shown to have Hh-responsive enhancer activity (Alexandre et al., above that expected by chance (see Materials and methods). By 1996; Forbes et al., 1993). At wg, a 2.5 kb element upstream of the probing with the consensus sequence (TGGGTGGTC), which transcription start site drives expression in metameric stripes represents an unknown fraction of Ci binding targets, we found that (Lessing and Nusse, 1998). This region has sequences that match its frequency in the DamCiAct and DamCiRep peaks was 2.4- to 2.5- the degenerate consensus Ci binding site (see Table S2 in the fold greater than expected by chance. Most consensus sequence supplementary material), and both DamCi forms conferred motifs were present in one copy only (e.g. motifs in 89% of nuclease protection. rdx requires Hh activity for expression (Kent DamCiAct peaks were singles) and their proximity to transcription et al., 2006), but its regulatory sequences have not been start sites was without apparent bias. We conclude that our current characterized. DamCi analysis revealed binding by both DamCi

understanding of Ci binding specificity leaves us without the means forms within rdx. Enhancers responsible for Hh regulation of ato DEVELOPMENT Targets of Hedgehog signaling RESEARCH ARTICLE 3891

Fig. 2. Expression array and DamID analyses. (A)Cluster analysis of expression arrays depicted as columns representing pairwise comparisons of transcript levels of da>CiAct, ptcB98/ptc6C, ci94/ci94, smoQ/smoQ and hhAC/hh13C to controls. Each lane represents one array experiment. Genes upregulated under normal or elevated Hh signaling are represented in rows as shades of green. bap, rdx, wg and en were previously shown to respond to Hh signaling in the embryo; hh, ato, CycE, drm and h have been shown to respond to Hh signaling in non-embryonic tissues. sna and ImpL2 are characterized in this study. (B)Above: Venn diagram indicating the number of genes linked to DamCiAct and DamCiRep sites or both. Below: Overlap between expression array targets and genes linked to DamID sites.

expression in eye discs and the PNS of the embryo have been were queried in this analysis); and one (lethal of scute) is expressed identified (Sun et al., 1998). The DamID-protected regions at ato in only six cells per hemi-segment and the levels of its transcripts spanned the identified enhancer elements. We interpret the might have been below detection. We suggest that the combination consistency of DamCiAct and DamCiRep binding, the presence of Ci of Ci binding sites revealed by DamID analysis, together with the consensus binding sites in DamCi protected regions and the tight response to Hh signaling revealed by the expression array analysis, correlation of DamID signal to known Hh targets as evidence that identifies a set of 52 probable Ci targets. In the following sections, this DamID analysis detected Ci binding sites that function in vivo. we describe our characterization of some of these genes as well as several others that are co-regulated. Hedgehog responsive genes To identify Hh-dependent transcripts, RNA extracted from five Tissue-specific responses to Ci types of stage 10-11 mutant embryos was compared with controls ptc and rdx are targets of Hh signaling that encode components of by hybridization to whole genome expression arrays. Mutant the Hh signal transduction pathway, and available evidence embryos for three positive regulators of the pathway (Hh, Smo and indicates that both are expressed in all cells that respond to Hh. By Ci) and one negative regulator (Ptc) were tested, as well as contrast, expression of all other Hh targets appears to be tissue- embryos in which CiAct was ectopically expressed (daGal4>CiAct). specific. Thirty known targets listed in Table 1 do not encode Clustering genes according to similarity of responses yielded a components of Hh signal transduction, and each has been shown to group of 147 (Fig. 2A). The abundance of transcripts from these be expressed in tissue-specific patterns. Our analysis of many of 147 genes was decreased in embryos with compromised Hh the new candidate targets indicates that they are also expressed in signaling (hh, smo, ci), and increased in embryos with elevated Hh only a subset of Hh-responsive tissues. Our analysis of candidate signaling (ptc, daGal4>CiAct). Mutants lacking the function of Hh target genes expressed in the embryo dorsal ectoderm, visual pathway components developed similar cuticular phenotypes, primordia and tracheal placodes is described below. therefore, this expression array analysis shows that the cuticular phenotypes have a common mechanistic basis in a failure to Dorsal ectoderm regulate the Hh signaling pathway and Hh target genes. Among the genes that are expressed in segmentally repeated stripes Genes in this group of 147 might be either direct or indirect in the embryo, three are members of the odd skipped family. These targets, and as wg is a direct target at each segment border of the genes, drumstick (drm), sister of odd and bowl (sob) and odd embryo, many of these Hh-responsive genes could be direct targets skipped (odd), are linked on chromosome 2L and are associated of Wg rather than Hh. Ci binding is presumably a property that with three regions of overlapping DamCiAct and DamCiRep binding distinguishes Hh targets, and 52 genes of the 147 had regions (Fig. 3A). Although drm is the only candidate that fulfills both the within, or adjacent to, their transcription units that were protected DamCi binding and cluster analysis criteria, odd and sob by DamCiAct or DamCiRep. Published work has shown that Hh expression changed in ways that are consistent with regulation by regulates 13 of the 30 known Hh targets in embryos, whereas the Hh signaling. drm, odd and sob were expressed in nearly identical others are targeted at later stages or are negatively regulated (Table patterns in stage 11 embryos (Fig. 3C-E). The stripes of drm, sob 1). Five of the thirteen embryo targets (atonal, bagpipe, engrailed, and odd expression were adjacent and posterior to hh-expressing rdx and wg) are among the 52 candidate genes. Of the seven that cells in each parasegment (Fig. 3C; data not shown), and are not, three (Drop, Wnt4, rhomboid) responded as expected of Hh expression in these domains is consistent with regulation by Hh. targets in some arrays but their responses in other arrays were We compared drm, sob and odd transcription in embryos that below our thresholds; four (ptc, huckebein, seven up, stripe) were express CiAct ubiquitously (daGal4>CiAct) with controls using ptc not included in the cluster analysis because their transcripts were expression as reference. In stage 11 embryos, ptc was expressed in not represented in the hybridization probes (presumably for failure many places, whereas in the trunk ectoderm, expression was

to amplify, meaning that only nine of the known embryo targets confined to two ectodermal stripes in each parasegment, one to DEVELOPMENT 3892 RESEARCH ARTICLE Development 137 (22)

Table 1. Hh target genes This study Previously identified Hh-dep Ci-dep Emb Reference Transcription factors asense* atonal* FF+ Dominguez et al., 1996 drumstick* araucan F Gomez-Skarmeta and Modolell, 1996 escargot* bagpipe* F + Azpiazu et al., 1996 fork head* caupolican FF Gomez-Skarmeta and Modolell, 1996 gooseberry* Drop F + D’Alessio and Frasch, 1996 knirps* engrailed* F + Bossing and Brand, 2006 lola* hairy* F + Hays et al., 1999 nerfin-1* huckebein F + McDonald and Doe, 1997 putzig* invected F Guillen et al., 1995 ribbon* knot FF+ Hersh and Carroll, 2005 sloppy paired 1* ladybird early f + Jagla et al., 1997 sloppy paired 2* ladybird late f + Jagla et al., 1997 snail* lethal of scute F + Bossing and Brand, 2006 worniu* seven up F + Ponzielli et al., 2002 stripe FF+ Piepenburg et al., 2000

Cell cycle regulators Cap-G* CycE* FF Duman-Scheel et al., 2002 CycA* CycD FF Duman-Scheel et al., 2002 miranda* Rac2* sticky*

Signaling pathways fat* dpp FF Dominguez et al., 1996 naked cuticle* hedgehog* FF Heberlein et al., 1995 Toll-6* ptc F + Alexandre et al., 1996 roadkill* F + Kent et al., 2006 rhomboid F + Alexandre et al., 1996 Serrate f + Alexandre et al., 1996 vein F Amin et al., 1999 wingless* FF+ Von Ohlen et al., 1997 Wnt4 F + Buratovich, 2000

Other and unknown CG6850* Cad99C FF Schlichting et al., 2005 CG1210* Cad86C FF Schlichting et al., 2005 CG3424* eyes absent F Pappu et al., 2003 CG4444* pxb f + Inaki et al., 2002 CG10176* CG10641* CG16815* CG8589* CG8414* CG13366* CG1677* CG6398* CG12702* CG8965* CG15628* Mes4*

Transcriptional or translational regulation bancal hnRNP* cactus* domino DNA dependent ATPase* mRNA capping enzyme* Hrb27C* trailer hitch* The current study identified 52 genes (*) based on two criteria: linkage with regions protected by DamCiAct and DamCiRep; and array clustering (Fig. 2A). These 52 genes, as well as previously identified targets (column 2), are listed and organized according to molecular function. For previously identified genes, published data regarding up (F) or down (f) regulation by Hh signaling or Ci activity and Hh- or Ci-dependence of expression in the embryo (+) are summarized in the adjacent columns. All previously identified genes are linked with DamCi-protected sequences, except for CycD, Cad99C and Cad86C. Hh-dep, regulation by Hedgehog signaling; Ci-dep, regulation by Cubitus interruptus activity; Emb, dependence on Hh or Ci in the embryo; DamCi, genes linked with DamCi- protected sequences. DEVELOPMENT Targets of Hedgehog signaling RESEARCH ARTICLE 3893

(not shown). The patterns of drm, sob and odd expression in wild- type and ubiquitous CiAct embryos are consistent with regulation by Hh and Ci-dependent activation specifically in the trunk ectoderm.

Visual primordium The visual primordium has anterior and posterior optic lobes (AOL and POL, respectively), and contains the precursor cells for Bolwig’s organ, the larval eye. During stage 11, the placode adopts a V-like shape and has conspicuous anterior and posterior lips. hh is expressed in the visual primordium and its expression pattern changes rapidly as the embryo matures. In situ hybridization and enhancer trap expression revealed that, at early stage 11, hh expression extended from the POL into the AOL, became restricted to the POL at mid stage 11 and later localized to Bolwig’s organ precursor cells in the ventral posterior lobe (Fig. 4A-C) (Chang et al., 2001). Fasciclin2 (Fas2) is expressed in the POL of stage 11 embryos where its expression coincides with hh (Fig. 4D,E) (Chang et al., 2001). Fas2 levels in the POL remained robust in hh mutants (Fig. 4F), indicating that Fas2 is not an Hh target and that at least some aspect of the POL remains in the absence of hh function. Full length Ci protein, a hallmark of active Hh signaling, was present in both the POL and adjacent AOL cells at mid stage 11 (Fig. 4G,H), and rdx expression, another hallmark of Hh signaling, had a similar pattern (Fig. 4L). smo was expressed in both lobes, although our in situ hybridization analysis was insufficient to distinguish relative levels of expression within the AOL and POL (Fig. 4I). By contrast, ptc RNA was clearly present only in AOL cells adjacent to the POL (Fig. 4J). Fig. 3. Genes in the odd skipped cluster are targets of Hh As elevated levels of rdx expression and full length Ci protein signaling in the dorsal ectoderm of Drosophila. (A)DamCiAct are hallmarks of paracrine Hh signaling, their presence in the POL Rep (orange vertical bars) and DamCi (blue vertical bars) signal at was unexpected. Hh-target genes are not expressed in hh- drumstick (drm), sister of odd and bowl (sob) and odd skipped (odd). expressing cells in the wing disc or embryo trunk ectoderm, a Red boxes indicate Ci motif clusters matching the motif YGSGDGGNC pattern that is attributed to repression by En. En also has a role in (see Table S1 in the supplementary material). (B-E)Wild-type expression patterns of ptc (B), drm (C), sob (D) and odd (E). Expression of ptc the positive regulation of hh expression, and loss of rdx expression coincides with the anterior margin of the tracheal pit (white line in B). in the ectodermal stripes of en mutant embryos is one of the Stripe of drm expression lies immediately posterior to hh-lacZ (brown consequences (Fig. 4N). However, En is not expressed in either staining in C). (F-I)Expression of drm (G), sob (H), and odd (I) expands optic lobe (Fig. 4K), and rdx expression remained robust in the posteriorly by two to three cell diameters in daGal4>UAS CiAct. ptc optic primordium of en mutant embryos (Fig. 4N). In situ expression expands similarly (F; arrow) and is activated in more cells of hybridization revealed that rdx RNA was reduced in both the AOL each parasegment. (J)Diagram of Hh target gene activation in the and POL of hh mutants (Fig. 4M), showing that hh function is dorsal ectoderm. ptc expression (tan) on the anterior and posterior essential for rdx expression in both regions. The POL is present in Act flanks of each hh stripe (blue) expands with ubiquitous Ci (right; red hh mutant embryos (Fig. 4F) and expression of rdx in cells dorsal stripes), but expression of Hh targets odd, drm and sob (brown), which to the POL was not affected by loss of hh (Fig. 4M). In wild type, in wild type (WT, left) is posterior to the dorsal portion of each hh stripe, expands only on the dorsal-posterior flank. a two to three cell-wide stripe of rdx expression connected with the dorsal part of the POL (Fig. 4L). This stripe extended dorsally and curved into the anterodorsal part of the head. A similar pattern of eyes absent (eya) expression was also observed (Fig. 5B), and for either side of each hh stripe (Hidalgo and Ingham, 1990). Levels both genes, this stripe was not altered in hh mutant embryos, of expression were higher in the more posterior stripe of each pair, consistent with a tissue-specific role for Hh signaling in the POL. and with ubiquitous CiAct, the general level of ptc expression These results indicate that hh expression in the optic primordium increased; the more intensely expressing posterior stripe broadened is not dependent upon En, and that Hh signal transduction in the posteriorly, and the more anterior stripe broadened anteriorly. These optic primordium is activated both in AOL cells (revealed by Ptc, changes are consistent with expansion of ptc expression to all cells Ci and rdx expression) and in hh-expressing POL cells (revealed except for those that express En. drm, sob and odd each responded by Ci and rdx expression). In the Discussion section, we consider to ubiquitous CiAct with posterior expansion of each stripe by two the possibility that hh-expressing cells of the POL respond to Hh to three cells (Fig. 3B-I). In wild-type germband-extended in an autocrine manner. embryos, drm and sob were also expressed in the presumptive We also monitored visual primordium expression of three genes hindgut and odd was expressed in the presumptive foregut. We did that are in the set of 2108 with linked DamCiAct and DamCiRep not observe changes to these domains of expression after binding sites: snail (sna), derailed (drl) and eya. They were ubiquitous expression of CiAct, nor did we observe ectopic examined because of their similarly strong upregulation in Act

expression in any other tissues in which Hh signaling is also active expression arrays of embryos with ubiquitous Ci . sna is one of DEVELOPMENT 3894 RESEARCH ARTICLE Development 137 (22)

Fig. 4. Hh signaling in Drosophila embryonic visual primordia. (A-N)Lips of the optic lobe (OL) placode are outlined with dotted lines. Anterior is to the left, dorsal is at the top. Patterns of hh expression in the Fig. 5. Novel Hh target genes in Drosophila embryonic visual OL change in early (A), mid (B) and late (C) stage 11 embryos. Fas2 primordia respond to CiAct in a tissue-specific manner. (A-I)Lateral expression (D; red) marks the posterior optic lobe (POL) and is coincident view of the visual primordia. Lips of the optic lobe (OL) placode are with hh-lacZ at mid-stage (E; green). Fas2 staining (F; red) marks POL in outlined with dotted lines. Anterior is to the left. In the wild type, drl hhAC (hh–). Full-length Ci (G; red) in the AOL and in Fas2-containing POL (A) and eya (B) are expressed in the POL. sna (C) is expressed in the (H; green). (I)smo expression in AOL and POL. (J)Ptc protein (arrow) in most dorsal cells (arrowheads) of both the AOL and POL. In the hh AOL. (K)En (red) is absent from the OL but is present elsewhere where hh mutant, POL expression of drl (D) is absent and expression of eya (E) is expressed (arrow). (L)rdx expression in POL, in adjacent AOL and in cells and sna (F) is reduced. In daGal4>CiAct mutants, expression of drl (G), dorsal to the OL (black arrow) and in ectodermal stripes (white arrows). eya (H) and sna (I) is unchanged. (J-O)Dorsal view of the visual (M)rdx expression in hhAC (hh–) is largely absent from the OL but is primordia. Expression of drl (J), eya (K) and sna (L) in the wild type is present in more dorsal cells (arrow). (N)rdx expression in enl11/en7 is shown. Note the absence of expression in the dorsal head ectoderm absent from ectodermal stripes but unaffected in the OL. (O)Diagram of (red dashed lines). drl (M) eya (N) and sna (O) expression in Hh target gene activation in the optic primordium. Left: Fas2 (green) in daGal4>CiAct fuses the normally bilateral fields of OL expression. the POL and Ptc protein in the adjacent AOL (brown). Middle: CiAct (red) in both the POL and adjacent AOL and Hh (blue) in the POL. Right: rdx (purple) in the POL and adjacent AOL. the 52 genes in the hh-dependent cluster; expression of the other primordium. In CiAct-expressing embryos, expression of all three two changed in ways that are consistent with regulation by Hh genes extended from the optic primordium into the dorsal ectoderm signaling, albeit at levels that did not meet the clustering thresholds. of the head (Fig. 5M-O). These dorsal domains of expression are In situ hybridization to stage 12 embryos revealed that sna, drl similar (or identical) to the Hh-induced cyclopia phenotype in ptc and eya were expressed in many tissues, and that in the optic mutant embryos (which have upregulated Hh signaling) and in primordium, sna was expressed in both lobes (Fig. 5C) whereas drl embryos that had been subjected to a heat shock-induced dose of and eya were expressed only in the POL (Fig. 5A,B). Expression ectopic Hh expression (Chang et al., 2001). of sna, drl and eya in the optic primordium of hh mutant embryos was severely reduced or undetectable (Fig. 5D-F). Whereas Tracheal placode expression of sna and eya in other tissues was unaffected in hh The tracheal system arises from twenty clusters that have mutants, the ectodermal stripes of drl were absent in mutant approximately 90 epidermal cells each. At stage 11, the tracheal embryos (not shown). With the exception of an increase in the pits have begun to invaginate, hh is prominently expressed in number of cells that express sna in the peripheral nervous system, posterior compartment epidermal cells to which the pits are expression of sna, drl and eya in embryos containing ubiquitous juxtaposed and Hh protein accumulates in the most anterior cells Act

Ci was affected only in cells associated with the optic of the pits (Glazer and Shilo, 2001). A role for Hh signaling in DEVELOPMENT Targets of Hedgehog signaling RESEARCH ARTICLE 3895 these anterior pit cells is indicated by their high levels of ptc expression and by the reduced number of cells in the pits of hh mutants (Glazer and Shilo, 2001). In situ hybridization studies confirmed that hh is expressed in the cells immediately anterior to the pits, and known targets of Hh, such as ci, ptc and rdx were expressed in the adjacent pit cells (Fig. 3; Fig. 6A-D). ptc was expressed most prominently in the anterior portion of the pits; ci was expressed more broadly and most pit cells had elevated levels of full-length Ci protein. Expression array analysis (Fig. 2A) and the DamID assay (Fig. 6Q) led to the identification of ImpL2 as an Hh target expressed in the tracheal pit. ImpL2 lacks a known function; its expression was activated in anterior cells of the pit (Fig. 6G) and in cells immediately dorsal to the pit invagination (Fig. 6M). It was expressed in only a subset of the cells in the tracheal primordium, which we defined by btl expression (Fig. 6J). Expression of ImpL2 was severely reduced but not absent in hh null embryos (Fig. 6H), suggesting that Hh signaling is necessary for activation of ImpL2, but that ImpL2 also responds to other activators. We monitored ImpL2 expression in embryos expressing ectopic CiAct to determine the maximum range of Hh-dependent activation. btlGal4>CiAct embryos had a higher number of ImpL2-expressing cells in the tracheal primordium (Fig. 6K), generating an expression pattern in the tracheal primordium that was indistinguishable from btl. ImpL2 expression expanded similarly to the entire primordium in daGal4>CiAct. ImpL2 expression dorsal to the tracheal primordium was not affected by btlGal4>CiAct (Fig. 6O), suggesting that these cells are not part of the tracheal primordium. Expression in this region is hh-dependent, as indicated by requirement for hh (Fig. 6H) and expansion in daGal4>CiAct (Fig. 6K). DamCiAct binds two regions at the ImpL2 locus; both contain Ci motifs (Fig. 6Q). One of these regions is in the first intron and contains three Ci motifs within a 285 bp sequence; adjacent to this cluster is a consensus Trh binding site (CTCGA). Trh is a basic helix-loop-helix (bHLH)-PAS DNA binding protein that has been implicated in tracheal development (Boube et al., 2000; Wilk et al., 1996). Our evidence suggests that Impl2 is a Trh target. In wild type, expression of trh was immediately dorsal to the tracheal pit invagination (Fig. 6E) and throughout the tracheal primordium. This pattern is similar to btl expression and to ImpL2 expression in Fig. 6. ImpL2 is a Hh target in the tracheal pit of Drosophila. Act btlGal4>Ci embryos. To determine if Trh function is necessary (A-P)Lateral views of stage 11 embryos. Anterior is to the left. hh (A) is for ImpL2 expression, we examined trh mutants. Although ImpL2 expressed in a stripe of cells immediately anterior to each tracheal pit expression in the cells dorsal to the tracheal primordium was (dashed circle; pit identified by gap). Pit cells express ci RNA (B), stabilize unaffected (Fig. 6N), it was essentially absent in the tracheal CiFL (C,D) and express rdx (D). trh is expressed in a broad region that primordium (Fig. 6I,N). Based on these observations, we conclude includes the pit (E) and trh expression in the pit is not diminished in AC that Trh and Ci together activate ImpL2 expression in the tracheal hh mutants (F). ImpL2 is non-uniformly expressed in the pit with highest levels in the most dorsal anterior cells (G). expression in pit. ImpL2 the pit is severely reduced in hhAC (H) and trh1/trh2 (I; arrow). Anti-GFP in a btl>GFP embryo reveals btl expression (J). ImpL2 expression in the DISCUSSION pit expands similarly in btl>CiAct (K) and da>CiAct (L). ImpL2 expression Because hh mutant embryos have an extensive syndrome of defects in ectodermal cells dorsal to the pit (M; arrow) present in trh mutants and hh is expressed in most organ systems in the embryo, we (N; arrow) and btl>CiAct (O; arrow), and elevated in da>CiAct (P; arrow). designed a global search for genes that are regulated by Hh to (Q)Statistically significant DamCiAct signal (orange bars) and Ci binding improve our understanding of its diverse roles. We used chromatin- motif clusters (red boxes) in first intron and downstream of ImpL2; binding assays to identify sequences that bind Ci in vivo, analyzed binding motif for Trh (green line) in first intron. Ci motif clusters match transcription profiles with expression arrays to identify genes the motif YGSGDGGNC and are listed in Table S2 in the supplementary whose expression is Hh-dependent, and monitored how several Hh- material. (R)Summary diagram of Hh target gene activation in the cells of the tracheal pit. Expression of (brown), (blue), Ci (red), target genes are expressed and regulated. These experiments hh ptc btl (gray), trh (light green) and Impl2 (dark green) in wild type (WT) and illuminate three general properties of Hh signaling: (1) targets of btl>CiAct mutants reveals the location of the tracheal pit relative to the Hh signaling include genes that control cell behavior directly (e.g. AP compartment border (the interface of ptc and hh expressing cells), cyclins) but a high proportion are transcription factors, with the extent of the tracheal primordium (defined by domains of btl and presumed indirect consequences; (2) Hh signal transduction is trh expression) and expansion of Impl2 to the entire tracheal Act activated in some Hh-expressing cells where it could be autocrine; primordium in the presence of ubiquitous Ci . DEVELOPMENT 3896 RESEARCH ARTICLE Development 137 (22) and (3) tissue-specific activation of targets requires the concluded that it is (Ramirez-Weber et al., 2000), implying that combinatorial activity of both Ci and non-Hh-dependent either Hh-expressing cells respond to Hh received from transcription factors. neighboring cells, or to Hh that they produce themselves. We do not know if the distinction between autocrine and Hh target genes paracrine signaling is important, but we are intrigued by the Chromatin binding and expression array analyses are, in essence, responsiveness of Hh-expressing cells to Hh in the optic fractionation methods and, like all such procedures, whether they primordium (Fig. 4). Cells in the POL express hh as well as rdx and are genetic screens, biochemical purifications, or behavioral have elevated levels of Ci, two hallmarks of pathway activation. selections, they are plagued with false positives and negatives. POL cells do not express detectable levels of Ptc, an Hh receptor Productive use of these methods therefore depends on additional whose negative regulation of the Hh signal transduction pathway means to extract candidates and determine relevance. is relieved upon Hh binding. Although loss of Ptc function can lead Chromatin-binding assays have a high frequency of false to ectopic pathway activation, expression of rdx in the POL appears positives, as they detect association with many more sites than are not to be a consequence of absent Ptc; rdx expression was likely to be functionally relevant. Whether binding to non- markedly reduced in hh mutant embryos, indicating that activation functional sites is an experimental artifact or represents the actual of the pathway in the POL is Hh-dependent. One possible distribution, it complicates identification of relevant targets. explanation is that some function other than Ptc keeps the Hh Statistical measures validate the binding assays, but they do not pathway in an ‘off’ state when Hh is not present. Another distinguish between the sites. Association with consensus binding possibility is that Ptc is expressed in these cells, but at levels too sequences might help, but binding to bona fide targets might not low for our methods to detect, and that, although pathway always be direct, and our understanding of how accessibility and activation increases ptc expression in other contexts (for instance, affinity affect residency is incomplete. It is especially poor for Ci in the AOL and in every known setting with paracrine signaling), and, although we had hoped that independent identification of hh-expressing cells of the POL keep ptc expression low. As the binding sites for CiAct and CiRep would establish whether they elevated level of Ci in the POL does not lead to high levels of ptc, target the same genes or not, our results were inconclusive. Many either the Ci is inert or POL cells have a mechanism to override Ci- of the protected DamID regions are shared and these overlaps are dependent activation. This mechanism cannot involve En, a statistically significant; moreover, we show that some of these negative regulator of ptc, because POL cells do not express en. regions are linked to legitimate targets. However, these chromatin In addition to our evidence for autocrine Hh signaling in the binding experiments cannot rule out the existence of functionally wing disc and optic primordium, apparent autocrine signaling by relevant sites that are specific to the activator or repressor forms. sonic hedgehog has been observed. Three examples are in neural Among the proteins encoded by these 52 targets, we identified six stem cells (Cai et al., 2008), large B-cell lymphoma (Singh et al., with roles in cell cycle regulation and nineteen that were 2010) and cerebellar dysplasia in the developing brain (Wang et al., transcription factors. Previous work has shown that Cyclin D and 2004). These examples indicate the potential importance of an Cyclin E are Hh targets in the eye disc, that Ci binds to and regulates autocrine mechanism to human development and disease. Cyclin E expression (Duman-Scheel et al., 2002), and that Cyclin E expression in the embryo is regulated in a tissue-specific manner Tissue-specificity of Hh-dependent responses (Jones et al., 2000). Our work adds Cyclin A and Sticky to the cell Activation of Hh targets dpp and knot in the wing disc requires cycle kinases regulated by Hh. The conceptual significance of these participation of Ci with Vestigial (Hepker et al., 1999) or Ataxin-2 observations is that Hh might promote cell division and growth Binding Protein 1 (Usha and Shashidhara, 2010), respectively. We directly by regulating such cell cycle functions. show here that activation of ImpL2 in the tracheal primordium In addition, the targets identified by our work and by previous requires Ci and Trh (Fig. 6). This supports the idea that cell studies show that Hh signaling also regulates cell behavior signaling pathways such as Hh are characterized by ‘activator indirectly. Hh controls growth and patterning of the wing disc by insufficiency’, meaning that pathway activation is necessary, but employing Ci to regulate Dpp expression at the anteroposterior not sufficient, to induce targets (Barolo and Posakony, 2002). compartment border (Tabata et al., 1995; Zecca et al., 1995). Ci In the tracheal primordium, our genetic analysis shows that, also upregulates Wg expression (Lessing and Nusse, 1998), as well although trh expression is not dependent on hh, ImpL2 expression as key components of the Notch and EGF signaling pathways depends on both hh and trh. These observations indicate that the (Table 1). Indirect regulation of cell behavior was also revealed by domain of trh expression must be established independently of Hh the high proportion of transcription factors among the targets: 19 and that ImpL2 responds only where both Hh signaling and trh out of 52 (37%). Transcription factors represent 40% (30 out of 74) expression intersect. As all cells in the tracheal primordium express of all targets of Drosophila Hh identified to date, and the trh but only a portion express ImpL2, the cells in which Hh prevalence of transcription factors is consistent with the recent signaling induces ImpL2 expression represent a subset of the cells tabulation of Shh targets in the vertebrate limb (Vokes et al., 2007). that are capable of responding. ImpL2 is expressed only in cells near Hh-producing cells that, presumably, are exposed to the Autocrine Hh signaling highest relative levels of Hh. Moreover, as ectopic activation of the Hh signaling is understood best in the wing disc and embryo, pathway led to upregulation of ImpL2 expression throughout the where posterior compartment cells that express En and Hh export tracheal primordium, the level of Hh signaling seems to be adjusted Hh to anterior compartment cells that express neither protein. in normal embryos to regulate ImpL2. Because ImpL2 expression Although the paracrine response of anterior cells is well- outside of the tracheal primordium is not trh-dependent, we established, posterior cells also activate Hh signaling (Ramirez- generalize these observations and suggest that the mechanism that Weber et al., 2000) but the basis for their pathway activation has leads to selective responsiveness of Hh targets involves both not been settled. Whereas one study concluded that pathway concentration-dependent responses as well as combinatorial control

activation is not Hh-dependent (Denef et al., 2000), another by Hh-sensitive and Hh-independent transcription factors. DEVELOPMENT Targets of Hedgehog signaling RESEARCH ARTICLE 3897

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